ABSTRACT Osteoporosis is the most common bone disease; 53.6 million Americans have a reduced bone mass, which increases their risk of fragility fractures. In addition to the high prevalence of vitamin D deficiency, recent studies show that approximately 60% of older men and 40% of older women have inadequate vitamin K intakes. Growing evidence indicates that there are important interrelationships between vitamin D and vitamin K on bone. Vitamin D increases intestinal calcium absorption and stimulates production of two proteins, osteocalcin and matrix Gla protein. Vitamin K plays an essential role in activating these proteins so that osteocalcin incorporates calcium in bone and matrix Gla protein inhibits calcification in soft tissues, including the kidney. Both vitamins D and K are important for optimal function of these proteins. Although vitamin D supplements are widely used to improve bone health, trials of supplemental vitamin D alone in reducing fractures showed inconsistent results. Emerging evidence indicates that supplemental D in the context of low vitamin K status is less effective on bone health measures. Therefore, low vitamin K status may account for some of the inconsistencies of trials testing vitamin D and bone health and may contribute to toxicities attributed to high-dose vitamin D such as kidney stones. No previous randomized controlled trials have been adequately powered to test effects of vitamin K status on fracture risk or the interaction of vitamin K status with high-dose, supplemental vitamin D on bone. To fill knowledge gaps, we propose an innovative, ancillary study to the large, NIH-sponsored, VITamin D and OmegA-3 TriaL (VITAL). VITAL is testing effects of supplemental vitamin D3 (cholecalciferol, 2000 IU/d), and/or omega-3 fatty acids (fish oil, 1 g/d) in the primary prevention of cancer and cardiovascular disease in 25,871 U.S. men (aged ?50) and women (aged ?55), including 5,106 African Americans. In this competitive renewal application of ?VITAL: Effects on Bone Structure and Architecture,? we will determine whether low vitamin K status, assessed by 3 sensitive biomarkers, modifies effects of supplemental vitamin D on fractures, bone mineral density (BMD) and structure, and secondarily on increases in urine calcium excretion, a risk factor for low BMD and kidney stones. During the first cycle of this grant, we surpassed recruitment goals and collected baseline and 2-yr post-randomization imaging studies for bone density, structure and architecture that will be used in the proposed studies (n=771). In addition, other key VITAL resources will be leveraged including: 16,953 baseline and 6,000 follow-up blood samples; measures of vitamin D, calcium, creatinine and parathyroid hormone; and adjudicated fractures and kidney stones. This proposal provides a unique opportunity to clarify in the largest study of supplemental vitamin D, the interdependencies of vitamins D and K and their roles on fractures, bone health measures, and urine calcium excretion. Findings from the proposed ancillary study will be critical to the interpretation of on-going vitamin D trials and provide new insights to advance clinical care and public health recommendations.